The present invention relates to a new protein and a nucleotide sequence encoding said protein. More precisely, the invention relates to a DNA molecule coding for a protein expressed by a bacterium of the genus Staphylococcus aureus, said protein and polypeptide fragments of said protein. Vectors comprising the nucleotide sequence coding for the protein, the protein and fragments thereof, and antibodies specifically binding to the protein may all be used for different vaccines against Staphylococcal infections in mammals. The invention also relates to a method of isolating and/or purifying apolipoprotein H from e.g serum with an immobilised protein or polypeptide of the invention.
Staphylococcus aureus is a pathogen responsible for a wide variety of diseases in humans and animals, including endocarditis, osteomyelitis, wound sepsis and mastitis. The bacterium produces several potential virulence factors such as alpha-, beta-, gamma- and delta-toxins, toxic shock syndrome toxin (TSST), enterotoxins, leucocidin, proteases, coagulase and clumping factor.
It is generally accepted that adhesion to tissues is required for bacterial colonisation to occur. For this purpose staphylococci express surface adhesins, which interact with host matrix proteins such as fibronectin, vitronectin, collagen, laminin and bone sialoprotein. In addition, staphylococci are able to bind several serum proteins, such as IgG, fibronectin, fibrinogen, and thrombospondin, possibly masking the bacteria from the immune system of the host. However, the contribution and importance of each of these binding functions in different infections is still unclear.
The most studied receptor in S. aureus is protein A, a cell wall-associated protein, which binds to the Fc- and the Fab-regions of IgG from several species. Protein A in strain 83254 consists of five consecutive, highly homologous domains, all with IgG-binding activity, followed by a region anchoring the protein in the cell wall (Uhlxc3xa9n et al, 1984). IgG-binding ability is common among clinical strains of S. aureus suggesting an important function in pathogenesis. It has been assumed that the IgG-binding capacity is mediated by protein A only.
However, the present inventors recently identified a nucleotide sequence in S. aureus strain 8325-4 encoding a polypeptide, clearly distinguishable from protein A, which binds IgG in a non-immune fashion (Jacobsson and Frykberg, 1995). An IgG-binding protein fragment having an amino acid sequence of 84 aa was disclosed. However, the amino acid sequence of the full length protein and the properties other than the IgG-binding ability of the 84 aa fragment were not known or even suggested. No nucleotide sequence coding for said protein has be disclosed or suggested prior to the present invention.
Diseases caused by Staphylococcal infections are often treated with antibiotics. As is well known in the art, these microorganisms can develop antibiotic resistance. Therefore, the use of vaccines to prevent or contain the spread of infection would be desirable. At present, there is no vaccine on the market that gives full protection. The present invention provides new immunologically active components for the production of vaccines against Staphylococcal infections.
The present invention is based on cloning and nucleotide sequence determination of a complete gene (sbi) encoding a novel IgG-binding protein. The gene encodes a protein of 436 amino acids, denoted protein Sbi, with one IgG-binding domain that exhibits an immunoglobulin-binding specificity similar to protein A and without the typical Gram-positive cell wall anchoring sequence LPXTG (SEQ ID NO:7) (Schneewind et al, 1995) suggesting that the protein is not anchored in the cell wall. Analysis of other S. aureus strains shows that this gene is not unique for strain 8325-4. For instance, the Sbi-protein is highly expressed in strain Newman 4, which shows that the IgG-binding activity observed in S. aureus is not mediated only by protein A. In fact, this (sbi) gene is present in all tested strains of S. aureus. 
Further, it has now been revealed that the Sbi protein of the invention binds apolipoprotein H, a major serum component, in addition to IgG. Hitherto, no bacterial protein binding to apolipoprotein H has been reported. Therefore, neither is this combination of the protein binding to these two serum components previously known. The portion of the protein which binds to IgG is located near the N- terminal of the protein, whereas the middle portion binds to apolipoprotein H. This enables the use of the protein, or an appropriate polypeptide fragment, in immobilised form for the isolation and/or purification of apolipoprotein H.
Thus, one aspect of the present invention is directed to a recombinant DNA molecule coding for a protein expressed by a bacterium of the genus Staphylococcus aureus, comprising the nucleotide sequence SEQ ID NO:1, defined in the sequence listing and the claims, or a homologous sequence to SEQ ID NO:1coding for said protein, or a partial or homologous sequence of the sequence SEQ ID NO:1 coding for a polypeptide fragment of said protein comprising at least 15 amino acid residues.
This recombinant DNA molecule may be inserted into plasmids, phages or phagemides for the expression/production of the protein or protein fragments.
Another aspect of the invention is directed to a protein expressed by a bacterium of the genus Staphylococcus aureus or a polypeptide fragment of said protein comprising at least 15 amino acid residues other than the 84 aa fragment at the position 38-121, which protein comprises the amino acid sequence SEQ ID NO:2, defined in the sequence listing and the claims, or a homologous sequence to the sequence SEQ ID NO:2 comprising a few mismatches in the amino acid sequence of SEQ ID NO:2, or polypeptide fragments of said homologous sequence comprising at least 15 amino acid residues.
The disclaimer of the 84 aa fragment at the position 38-121 of the SEQ ID NO:2 is made because, as already mentioned, it has been previously disclosed (Jacobsson and Frykberg, 1995).
It is well known in the art that there may be a few mismatches of amino acids residues in the amino acid sequence of a protein while the protein still retains its major characteristics. The mismatches may be replacements of one or several amino acids, deletions of amino acid residues or truncations of the protein. Such mismatches occur frequently in genetic variations of native proteins. It is believed that up to 15% of the amino acid residues may be replaced in a protein while the protein still retains its major characteristics. The protein of the invention comprises 436 amino acid residues, and therefore up to 66 mismatches would be acceptable. However, preferably there will be less than 20, more preferably less than 10, and most preferably less than 5 mitsmatches in the amino acid sequence of the protein of the invention.
The polypeptide fragments of the protein of the invention should comprise at least 15 amino acid residues to be sure that the fragments are not found in other known proteins. These fragments may be used e.g. as probes, diagnostic antigens, and vaccine components, possibly coupled to carriers.
In an embodiment of this aspect of the invention a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:3, defined in the sequence listing and the claims. This polypeptide fragment lacks the signal sequence of the SEQ ID NO:1.
In another embodiment a polypeptide fragment of the protein according to the invention has an amino acid sequence SEQ ID NO:4, defined in the sequence listing and the claims. This polypeptide fragment binds apolipoprotein H.
In yet another embodiment a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:5, defined in the sequence listing and the claims. This 120 aa polypeptide fragment binds IgG. It was chosen for immunisation purposes, in stead of the known IgG binding 84 aa fragment, since once the whole amino acid sequence was deduced, it became evident there were sequence similarities suggesting two IgG binding domains.
In still another embodiment a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:6. This polypeptide fragment binds apolipoprotein H, and has been used for isolation and purification of said serum protein.
In a preferred embodiment of this aspect of the invention the protein or polypeptide according to the invention is coupled to an inert carrier or matrix. The carrier may be e.g. plastic surfaces, such as microplates, beads etc.; organic molecules such as biotin; proteins, such as bovine serum albumin; peptide linkers, polypeptides e.g. resulting in fusion proteins. The matrix may be particles used for chromatographic purposes, such as Sepharose(copyright).
A further aspect of the invention is directed to a vector selected from the group consisting of plasmids, phages or phagemides comprising a nucleotide sequence according to the invention.
These vectors may be used for the production of the proteins or polypeptides of the invention. They may also be used in vaccines.
Yet another aspect of the invention is directed to antibodies specifically binding to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4, and SEQ ID NO:6. The specific binding of binding of an antibody to an amino acid sequence of the invention requires e.g an affinity constant of at least 107 liters/mole, preferably at least 109 liters/mole.
The antibodies of the invention may be monoclonal or polyclonal. They may be used in diagnostic tests, but preferably in vaccines for passive immunization.
Still another aspect of the invention is directed to the use of a protein or polypeptide according to the invention, optionally in immobilised form, as an immunising component in the production of a vaccine against Staphylococcus infections.
Another use aspect of the invention is directed to the use of a vector according to the invention for the production of a vaccine against Staphylococcal infections.
Yet another use aspect of the invention is directed to the use of antibodies according to the invention for the production of a vaccine for the passive immunisation of a mammal against Staphylococcus infections.
An additional aspect of the invention is directed to a vaccine against Staphylococcal infections comprising as an immunising component a protein or polypeptide according to the invention, optionally in immobilised form.
Another vaccine aspect of the invention is directed to a vaccine against Staphylococcal infections comprising a vector according to the invention.
A DNA molecule, or the corresponding RNA derived from the present sequence, as described in claim 1, may be used in a vector for vaccine purposes. Examples of suitable forms of administration include intravenous, percutaneous, and intramuscular administration.
Yet another vaccine aspect of the invention is directed to a vaccine for the passive immunisation of a mammal, especially a human being, against Staphylococcus infections comprising antibodies according to the invention.
One embodiment of the invention comprises the passive immunization of patients with an impaired immune defense or patient awaiting major surgery, such as patients in line for an organ transplantation or awaiting the insertion of a prosthetic device, such as a hip prosthesis or similar major surgical intervention. According to the present invention, a high dose of antibodies against the novel protein can be administered to any patient before or at the time of hospitalisation, in order to prevent Staphylococcus infection.
The vaccines may contain other ingredients selected with regard to the intended administration rout, and these ingredients are chosen by the vaccine manufacturer in collabaration with pharmacologists. Examples of administration routs include intravenous administration, percutaneous administration, oral and nasal administration.
A further aspect of the invention is directed to a method of prophylactic and/or therapeutic treatment of Staphylococcus infections in a mammal comprising administration to said mammal of an immunologically effective amount of a vaccine according to any one of the vaccines of the invention.
Still another aspect of the invention is directed to a method of isolating and/or purifying apolipoprotein H from a liquid medium, especially from serum, comprising chromatographic separation of apolipoprotein H from said liquid medium with an immobilised protein or polypeptide according to the invention as stationary phase.
In a preferred embodiment of the invention column chromatography is used for the isolation/purification of apolipoprotein H from blood serum. The protein or polypeptide of the invention is coupled to e.g. Sepharose(copyright) and is used as packing material for the column. The apolipoprotein H-containing serum is brought into contact with the immobilized protein or polypeptide and the apolipoprotein H is adsorbed. Finally, the apolipoprotein H is eluated from the column.